How in the heck did the deer get the copper in them? This is vet study.

"Necropsy of 2 white-tailed deer fawns who died acutely revealed diarrhea and melena in case No. 1 and no gross changes in case No. 2. Histologically, the livers of both deer displayed multifocal coagulative necrosis, with infiltrations of neutrophils, macrophages, and lymphocytes. By Warthin-Starry staining, bundles of filamentous bacteria were identified within hepatocytes at the periphery of the necrotic foci in case No. 1."

Related to myocarditis which is end stage in Chagas Disease, but with the modified trypanasoma protist added into the construct. Also an association to copper. Copper is an allergen to many mammals and humans. But, in this case is toxic.

I am going backwards, since forward thinking people only see the road ahead and cannot remember where they went.

from the cited journal for the experiment on the infected mice which is an experiment with interluken which is a study on immunity.

Now, if animals are getting a hepatic type disease, tell where that might come from?

I look at the references also.

But, what is not told the public from the health departments is that this is happening to animals. And it does not happen to humans?

Why are they making a study of this, if there is no danger to mammals or humans?

It is why and what that is not reported to the public.

Biofilms are forming in animals, why? The study on mice is to find the immune problem, to vaccinate.

If one realizes that it is the bacteria, quorem sensing and signalling that sets the stage for this. Too much bacteria in air, water, and plants.

to beat the local devastation, one has to start locally, not top down, that is what the CDC does. Each area is different, with different novel organisms thrown in the environment, just to see what it will do and to control all aspects of every living biological entity.

It is power at the top that is infiltrating locally in ways in plain sight, that no one questions. They are called agents, or NGO's or agents of the environmental control of every organism on earth, a new tree of life, because the one we have isn't good enough because it does not include the new novel organisms.

This is why animals are getting sick. Many of them.

We have CJD in deer here, add hepatitis to that and toxic copper.

Where is it coming from? and it is not in cattle? or horses?
or cats or dogs? The animals have been suffering for some time now.

"Tyzzer's disease is caused by the obligate intracellular bacterium called Clostridium piliforme (formerly Bacillus piliformis). The microbe was discovered and characterized in 1917 by Ernest Edward Tyzzer, a professor at Harvard Medical School and an authority on avian coccidia. He recovered C. piliforme from Japanese waltzing mice (Mus bactrianus) and published his findings in a comprehensive 37-page article1. Tyzzer provided a very thorough characterization of the agent, including clinical aspects of the disease, epidemiology, pathology, and reproduction of disease after experimental inoculation by several routes or exposure to soiled bedding. Bedding that was held for over one year remained infectious for mice.

The host range of C. piliforme is broad and includes all laboratory rodents and rabbits, several wild rodents, domestic animals, and several exotic non-rodent hosts. Much of our knowledge about host range is derived from case reports. One common theme is that the animals involved were frequently very young (puppies, kittens, calves, foals) or were immunosuppressed by virtue of another, often viral, infection2-5.

The target organs in animal infections are the intestine (distal ileum, cecum and proximal colon), the liver, and less often, the heart1. Enteric lesions can be severe, often with demonstration of bacilli in epithelial cells and smooth muscle cells of the muscularis mucosa and tunica muscularis interna and externa. The most frequently encountered lesion in all species of affected animals is focal hepatic necrosis. Since the first description of Tyzzer's disease in 1917, there has been only a single case report documenting a C. piliforme infection of a human being6. As was true of the animal case reports, the individual was severely immunosuppressed due to an intercurrent viral infection; however, the single human infection presented as skin lesions rather than the internal lesions described for other species,",,,,,,,

"The organism causing Tyzzer's disease is unusual. It is intracellular, pleomorphic, spore-forming, and motile. Although gram stains indicate that C. piliforme is gram-negative, the organism falls into a category of agents that are "extremely oxygen-sensitive (eos)" and can be shown to be gram-positive only if fixation and staining are done under anaerobic conditions7,8. C. piliforme stains weakly with hematoxylin and eosin, so confirmation is frequently made using silver impregnation or Giemsa stains. The vegetative form, readily visible microscopically, is large and ranges from 8-40 mm in length. The organism does not grow in cell-free medium, but does replicate in yolk sacs of embryonic eggs and in certain cell cultures. Vaccine-challenge studies have supported the hypothesis that C. piliforme isolates are morphologically identical but antigenically distinct9,10.

Transmission of C. piliforme occurs primarily by ingestion of spores in contaminated feces. The vegetative form is labile and is not thought to play a significant role in natural transmission. The spores, however, may remain infectious for years......

The diagnosis of Tyzzer's disease most frequently hinges on light microscopic observation and/or serologic detection. Seroconversion relies on active replication of viable organisms present within body tissues. Gross liver lesions are not always present. Immunosuppression of suspect colonies, in attempts to induce clinical disease, has met with variable success. And gerbils, although very susceptible to many C. piliforme isolates, are not universally susceptible.

Possible sources of C. piliforme include bedding, feed, and water. Antibiotic treatment simply drives the organism "underground," reducing obvious clinical disease. Decontamination is difficult because of the spore-forming nature of the bacterium; however, spore inactivation can be achieved by autoclaving supplies and/or by the use of gamma irradiation or ethylene oxide. Treatment with 0.3% sodium hypochlorite or 2% peracetic acid for 5 minutes also inactivates the spores18. C. piliforme spores are resistant to ethanol, phenolic germicidal detergents, and quaternary ammonium compounds and show a graded response to formaldehyde based on contact time18."

Honey, you must be scarrrrrred; scared of the truth. I can prove every one of my gd articles. Want me to????? b/c I'd love it......but....although I'm holding out for maybe a cure or some dough....

looks like I might just have to give in an nail ya......and.....you KNOW I CAN!!!!! One more comment from you witch and I begin. I will start with Oklahoma State University and will take you all the way to the netherlands and end up in your backyard.......Virginia///////

Ready???? Just say when.

All you do is lie.......and the people you want people to write to are nothing more than part of the cover up.......I mean even your honorable judge connection is........uhhh, well, he's heavily invested in the biotech field is HE NOT??????

I'm all for anyone making the dough but don't come on here and scream that we are the ones that are crazy.....only makes you look real......a witch that lies////////and lies and changes her story,,,,,,,,,,,,,,,,you are stressed out honey and it shows.......

you should go smoke some ricin or something.......now get on that broom and jet away............................
***************************************

Skytroll,

I'm very sorry, I just read your post but have not finished the links yet.
I don't think we should waste our time here to tell you the truth......

Sky, did you find the CdC yet.??????heheheheheheheheeeee Did you read where I said the microbes were.....hehehehe I will go ahead and post that link I guess since I'm so paranoid.......

but here. First read what molecular technology is......

Projections
2.2 What is molecular nanotechnology?
Molecular nanotechnology is an anticipated manufacturing technology that will make it possible to build complex three-dimensional structures to atomic specification using chemical reactions directed by nonbiological machinery. In molecular manufacturing, each atom would go to a selected place, bonding with other atoms in a precisely designated manner. Nanotechnology promises to give us thorough control of the structure of matter.

Since most of the stuff around us and inside us is composed of atoms and gets its characteristic properties from the placement of these atoms, the ability to control the structure of matter on the atomic scale has many applications. As K. Eric Drexler wrote in Engines of Creation, the first book on nanotechnology (published in 1986):

Coal and diamonds, sand and computer chips, cancer and healthy tissue: throughout history, variations in the arrangement of atoms have distinguished the cheap from the cherished, the diseased from the healthy. Arranged one way, atoms make up soil, air, and water arranged another, they make up ripe strawberries. Arranged one way, they make up homes and fresh air; arranged another, they make up ash and smoke.

Nanotechnology, by making it possible to rearrange atoms effectively, will enable us to transform coal into diamonds, sand into supercomputers, and to remove pollution from the air and tumors from healthy tissue.

Central to Drexler’s vision of nanotechnology is the concept of the assembler. An assembler would be a molecular construction device. It would have one or more submicroscopic robotic arms under computer control. The arms would be capable of holding and placing reactive compounds so as to positionally control the precise location at which a chemical reaction takes place. The assembler arms would grab a molecule (but not necessarily individual atoms) and add it to a work-piece, constructing an atomically precise object step by step. An advanced assembler would be able to make almost any chemically stable structure. In particular, it would be able to make a copy of itself. Since assemblers could replicate themselves, they would be easy to produce in large quantities.

There is a biological parallel to the assembler: the ribosome. Ribosomes are the tiny construction machines (a few thousand cubic nanometers big) in our cells that manufacture all the proteins used in all living things on Earth. They do this by assembling amino acids, one by one, into precisely determined sequences. These structures then fold up to form a protein. The blueprint that specifies the order of amino acids, and thus indirectly the final shape of the protein, is called messenger RNA. The messenger RNA is in turned determined by our DNA, which can be viewed (somewhat simplistically) as an instruction tape for protein synthesis. Nanotechnology will generalize the ability of ribosomes so that virtually any chemically stable structure can be built, including devices and materials that resemble nothing in nature.

Mature nanotechnology will transform manufacturing into a software problem. To build something, all you will need is a detailed design of the object you want to make and a sequence of instructions for its construction. Rare or expensive raw materials are generally unnecessary; the atoms required for the construction of most kinds of nanotech devices exist in abundance in nature. Dirt, for example, is full of useful atoms.

By working in large teams, assemblers and more specialized nanomachines will be able to build large objects quickly. Consequently, while nanomachines may have features on the scale of a billionth of a meter – a nanometer – the products could be as big as space vehicles or even, in a more distant future, the size of planets.

Because assemblers will be able to copy themselves, nanotech products will have low marginal production costs – perhaps on the same order as familiar commodities from nature’s own self-reproducing molecular machinery such as firewood, hay, or potatoes. By ensuring that each atom is properly placed, assemblers would manufacture products of high quality and reliability. Leftover molecules would be subject to this strict control, making the manufacturing process extremely clean.

The speed with which designs and instruction lists for making useful objects can be developed will determine the speed of progress after the creation of the first full-blown assembler. Powerful software for molecular modeling and design will accelerate development, possibly assisted by specialized engineering AI. Another accessory that might be especially useful in the early stages after the assembler-breakthrough is the disassembler, a device that can disassemble an object while creating a three-dimensional map of its molecular configuration. Working in concert with an assembler, it could function as a kind of 3D Xerox machine: a device for making atomically exact replicas of almost any existing solid object within reach.

Molecular nanotechnology will ultimately make it possible to construct compact computing systems performing at least 1021 operations per second; machine parts of any size made of nearly flawless diamond; cell-repair machines that can enter cells and repair most kinds of damage, in all likelihood including frostbite [see “ REF _Ref50109542 \h What is cryonics? Isn’t the probability of success too small?”]; personal manufacturing and recycling appliances; and automated production systems that can double capital stock in a few hours or less. It is also likely to make uploading possible [see “What is uploading?”].

A key challenge in realizing these prospects is the bootstrap problem: how to build the first assembler. There are several promising routes. One is to improve current proximal probe technology. An atomic force microscope can drag individual atoms along a surface. Two physicists at IBM Almaden Labs in California illustrated this in 1989 when they used such a microscope to arrange 35 xenon atoms to spell out the trademark “I-B-M”, creating the world’s smallest logo. Future proximal probes might have more degrees of freedom and the ability to pick up and deposit reactive compounds in a controlled fashion.

Another route to the first assembler is synthetic chemistry. Cleverly designed chemical building blocks might be made to self-assemble in solution phase into machine parts. Final assembly of these parts might then be made with a proximal probe.

Yet another route is biochemistry. It might be possible to use ribosomes to make assemblers of more generic capabilities. Many biomolecules have properties that might be explored in the early phases of nanotechnology. For example, interesting structures, such as branches, loops, and cubes, have been made by DNA. DNA could also serve as a “tag” on other molecules, causing them to bind only to designated compounds displaying a complementary tag, thus providing a degree of control over what molecular complexes will form in a solution.

Combinations of these approaches are of course also possible. The fact that there are multiple promising routes adds to the likelihood that success will eventually be attained.

That assemblers of general capabilities are consistent with the laws of chemistry was shown by Drexler in his technical book Nanosystems in 1992. This book also established some lower bounds on the capabilities of mature nanotechnology. Medical applications of nanotechnology were first explored in detail by Robert A. Freitas Jr. in his monumental work Nanomedicine, the first volume of which came out in 1999. Today, nanotech is a hot research field. The U.S. government spent more than 600 million dollars on its National Nanotechnology Initiative in 2002. Other countries have similar programs, and private investment is ample. However, only a small part of the funding goes to projects of direct relevance to the development of assembler-based nanotechnology; most of it is for more humdrum, near-term objectives.

While it seems fairly well established that molecular nanotechnology is in principle possible, it is harder to determine how long it will take to develop. A common guess among the cognoscenti is that the first assembler may be built around the year 2018, give or take a decade, but there is large scope for diverging opinion on the upper side of that estimate.

Because the ramifications of nanotechnology are immense, it is imperative that serious thought be given to this topic now. If nanotechnology were to be abused the consequences could be devastating. Society needs to prepare for the assembler breakthrough and do advance planning to minimize the risks associated with it [see e.g. “Aren’t these future technologies very risky? Could they even cause our extinction?”]. Several organizations are working to preparing the world for nanotechnology, the oldest and largest being the Foresight Institute.

SKYTROLL, THE DOE??????? THIS IS WHAT IT'S MAINLY ABOUT.....THEY CAN USE US AS BATTERIES.///////

I hope I'm wrong and you are right......but......I feel quite confident that they want to send us to Venus by 2007...you know,,,,,human trial runs first.....hey, go back 4 OR 5 PAGES AND YOU WILL SEE WHERE i POSTED AND ARTIClE on this with a hyperlink........Hey hehehe, I just saw your "better in pictures" post and LOVED IT...Skytroll, you should get tested and a lot of us should to on this,,,,,,,I don't underestimate it at all.
Before I post it, I will say that it is spread via the water.....

OMG, I just remembered. About 4 months ago, I found some invoice reciepts online from Morg research and guess where it led me too( of course they were coded but....cha ching, I got thru.....They led me to sanitation and water/sewer treatment plants.....I swear to God.

It was previously reported that the gag proteins of mammalian type C retroviruses are modified by the addition of myristate to the N-terminal glycine residue. We have performed oligonucleotide-directed mutagenesis to change this glycine codon in the Moloney murine leukemia virus genome to an alanine codon and also to specifically delete the glycine codon. Upon transfection into mammalian cells, these mutant genomes direct the synthesis of gag proteins, but these proteins are not myristylated. The mutants do not form virus particles or any recognizable virus-specific structures visible in thin sections with the electron microscope. Further, the mutant gag proteins appear to remain in the cytosol, whereas the wild type is found principally in particulate fractions of the cell. The results are consistent with the theory that myristate is required for the association of the gag protein with the plasma membrane and that this association is necessary for virus assembly.

Dear Reliefseeker -- As a nurse I have to ask, have you had cellulitis before and are you on any med to treat the concomitant CHF? Cellulitis can develop from venous insufficency, or from a fungal or bacterial infection. Have the doctors told you which came first -- the CHF or the cellulitis. Other diseases that may sometimes mimic cellulitis include contact dermatitis, drug or foreign body reactions, urticaria, lymphedema, lupus erythematosus, sarcoidosis, lymphoma, leukemia, paget disease, dendritic cell sarcoma, rheumatoid arthritis, and panniculitis. The most common symptoms of cellulitis are redness of the skin, swelling, warmness, pain or tenderness, drainage or leaking of yellow clear fluid or pus from the skin. If you have draining blisters, keep them covered until a scab forms. Leave the scab on to complete healing. It is normal for itching to increase more as skin heals, and one reason is because of the release of histamine which aids healing. I suggest looking at some cellulitis resources. If a doctor wants to release you back to work, they need reminded of that fact that you keep your leg unprotected and steadily exposed. Ativan is a drug that's used to relieve anxiety, nervousness, and tension associated with anxiety disorders. It is also used to treat certain types of seizure disorders and to relieve insomnia. Actually, 'atarax' is a good prescription antihistamine that helps with itching and has a mild sedating effect. That could help you rest better too, but it sounds like pain med might be most of what you need to help you allow your leg to heal.

Additional reminder for all of us -- Despite of all the trouble that sebum causes, it is very important to the maintenance of skin. When the system is working properly, sebum performs the important job of helping to lubricate skin. Sebum also carries with it dead skin cells shedding from our hair follicle walls. Any type of clay or drying product will coat sebum plugs and that can look confusing. Sebum keeps skin from drying out, but on the other hand, if a body is out of balance, oily skin tends to be a magnet for more dirt, dust and internal and external wastes than dry skin. Dry skin carries its own set of problems and is common as we age. But when skin is injured sebum and plasma cells leak out and form a grannular film -- that should be left in place to protect and heal, as it is rich with cells we need.

The purpose of my research over the past 30 years has been to isolate and characterize embryo-defective (emb) mutants of Arabidopsis. My initial goal was to use selected mutants to gain insights into the genetic control of plant embryo development. This objective was realized in part through the analysis of mutants with alterations in cotyledon and suspensor cell identity. Other mutants provided valuable clues to basic questions in plant metabolism, physiology, and cell biology. Extensive linkage analysis of EMB genes played an important role in enhancing the classical genetic map. With continued advances in Arabidopsis genomics, my research expanded to include the global identification of essential genes. This effort was made possible through an extended collaboration with John McElver and David Patton at Syngenta involving the large-scale identification of EMB genes through forward genetic screens of T-DNA insertion lines. This work provided the foundation for the Arabidopsis SeedGenes Project, which aims to present detailed information on all known genes with a knockout phenotype in the seed. The project database (http://www.seedgenes.org), constructed in collaboration with Allan Dickerman (Virginia Bioinformatics Institute), includes information on more than 500 mutants and 300 different genes. The frequency of mutants obtained to date is consistent with 500 to 1000 total EMB genes in Arabidopsis. A major challenge for the future is to find those genes that have so far escaped detection. We have identified several classes of candidate genes that merit further analysis through reverse genetics, including those that resemble essential genes in other organisms, those for which a knockout homozygote cannot be identified, and those that share an interacting protein, metabolic pathway, or cellular process with a known EMB gene. The ultimate goal is to identify and understand all non-redundant genes required for Arabidopsis seed development.

and Skytroll, here is a whole page where you can order the riber optic recievers....never know when we will need to replenish our stock.....thanks mouser elelctronics!

laboratories that implement this approach will benefit by the improved analytical performance and ease of use to conduct trace residue analysis of pesticides in food.

Development and evaluation of the “solvent in silicone tube extraction” (SiSTEx) approach. Effective and efficient analytical methods are needed to detect pesticide residues in fruits and vegetables. Rapid methods usually do not detect very low amounts of the pesticides because it takes time to concentrate the residues in a final extract. Furthermore, other chemicals in the food sample are also concentrated to the same extent as the pesticides in traditional methods, which interfere in the detection. This research study evaluated a new way to concentrate the pesticides, but block the interfering chemicals. The new approach is called “solvent in silicone tube extraction” (SiSTEx), in which an organic solvent is placed in a thin silicone tube surrounded by the watery sample. Many of the pesticides transfer into and through the tubing into the solvent, and most of the other chemicals stay in the watery sample. The pesticides are more highly concentrated in the solvent and very low levels can be detected from the sample. This new approach is simple, cheap, and effective, and can be used to lower detection limits of pesticide residues in food samples.

Further evaluation of low-pressure gas chromatography-mass spectrometry. Current methods of analysis for pesticide residues and other chemical contaminants in food are time-consuming. Low-pressure gas chromatography/mass spectrometry (LP-GC/MS) is a unique approach to speed the analysis of many types of chemical residues, including pesticides. A comparison was made between a wider analytical column of thicker film versus a narrower column of thinner film in LP-GC/MS. The narrower column configuration gave a slightly faster separation of the pesticides, but the wider analytical column with a thicker film gave and greater sensitivity and ruggedness. The analysis of 57 pesticides was optimized and demonstrated using LP-GC/MS with separation time less than 6 min, which is 5-7 times faster than traditional methods of analysis. This form of fast-GC/MS approach shows promise to become a widely used tool to help increase sample throughput in the analysis of pesticide residues.

Multiresidue quantitation and confirmation of fluoroquinolones in shrimp. The liquid chromatography-fluorescence-mass spectrometryn method developed for use in chicken muscle, liver and eggs was applied successfully, after minor modification, to shrimp samples fortified with 8 fluoroquinolones, as well as samples incurred with enrofloxacin. This project was a collaborative effort with Luz Vazquez-Moreno at CIAD in Sonora, Mexico. The method would be suitable for CIAD’s efforts in monitoring shrimp samples for fluoroquinolones residues.

Distribution of enrofloxacin residues in incurred chicken breast vs thigh muscle. When a tolerance level is established for veterinary drug residues in animal tissue, such as muscle, the type of muscle is typically not specified (e.g., breast vs. thigh). There is potential for drug residues to preferentially distribute within a selected type of muscle tissues. In this collaborative study with Dan Donoghue at the University of Arkansas, the liquid chromatography-fluorescence-mass spectrometryn method was used to supplement bioassay data in an effort to adddress this question. In this study, higher enrofloxacin concentrations were found in breast muscle samples, as compared to thigh muscle samples, after dosing of chickens with the drug. These types of studies can thus provide helpful guidance to those involved in setting tolerance levels for drugs in animal tissues.

Progress report.
This report serves to document research conducted under a specific cooperative agreement between ARS and the Dept. of Biosciences and Biotechnology at Drexel University, entitled “Development of New Methods to Detect and Control Acrylamide Formation in Deep-Fat Fried Foods”. Additional details of research can be found in the report for the parent CRIS 1935-42000-044-00D, “Advanced Techniques for the Analysis of Chemical Residues in Foods” The objective of this cooperative research project is to develop a new analytical method for acrylamide in foods that is faster, easier, and cheaper than the current methods, and to use this method for monitoring acrylamide in deep-fat fried foods in which different frying techniques will be assessed in order to minimize the generation of acrylamide in the food.

Acrylamide has become an important food safety concern, especially for regulators and the food industry, since the report of its presence in several types of processed foods in 2002, and effective methods are needed for its efficient analysis by many laboratories worldwide. In 2004, ARS developed a new rapid method using the QuEChERS concept, and optimized and evaluated the method in proficiency test samples in which results compared favorably versus other methods in use. The new, highly streamlined method uses only a single vessel in an easy liquid-liquid partitioning extraction and dispersive solid-phase cleanup approach for a variety of food types followed by LC/MS-MS analysis. Acrylamide mitigation strategies are being pursued by Dr. Phil Handel at Drexel University and ARS is analyzing the generated samples. Furthermore, Dr. Xuetong Fan of ARS is studying the effect of irradiation as another mitigation possibility. If the presence of acrylamide in processed foods can be avoided, this would solve a food safety dilemma and controversy surrounding the issue.

This report serves to document research conducted under U.S.-Israel Binational Agricultural Research and Development (BARD) grant US-35000-03 shared equally between ARS and the the School of Chemistry at Tel Aviv University in Tel Aviv, Israel, entitled “Fast, Practical and Effective Approach for the Analysis of Hazardous Chemicals in the Food Supply”. Additional details of research can be found in the report for the parent CRIS 1935-42000-044-00D, “Advanced Techniques for the Analysis of Chemical Residues in Foods” Dr. Lehotay collaborates with Prof. Aviv Amirav of Tel Aviv University on the 3-year grant. The ARS objectives of the project are to: A) expand the QuEChERS approach to veterinary drugs and environmental pollutants; and B) integrate automated DSI into the QuEChERS approach for GC/MS analysis. For Tel Aviv Universtiy, the objectives include: A) develop fast methods for difficult analytes using Supersonic GC/MS; and B) develop a Supersonic LC/MS instrument for the analysis of polar and labile analytes. In the final year of the grant, the goals of both collaborators will be to: A) combine the expanded, automated QuEChERS approach with automated DSI + fast Supersonic GC/MS and Supersonic LC/MS analysis; and B) evaluate the expanded, automated QuEChERS approach for real samples. In 2005, 2 automated DSI devices were purchased from competing vendors for evaluation to meet ARS objectives A and B. Arrangements have been made for a postdoc to conduct the research objectives described in the project. Initial experiments have been conducted pertaining to sample preparation in veterinary drug analysis, which show the QuEChERS approach for pesticides to be feasible after modifications depending on the matrix and analytes. Objectives A and B for Tel Aviv University have already been demonstrated for certain difficult pesticides and drugs. Good progress is also being made by Prof. Amirav toward making continual improvements in the prototype Supersonic GC/MS and LC/MS instruments used in his laboratory.

... . you gotta love this.. . . .. .

Improve Microbiological Safety and shelf-life of food by Treatment with ionizing Radiation

Furan, a toxicant, is listed as reasonably anticipated to be a human carcinogen by the U.S. Department of Health and Human Services. A recent survey by the Food and Drug Administration (FDA) revealed furan is present in a wide variety of foods that undergo heat treatment. This study was conducted to investigate possible formation of furan in fruit juices as a result of ionizing radiation, a non-thermal processing technology used for enhancement of food safety and extension of shelf-life. Our results showed that irradiation and thermal treatments induced furan in both apple and orange juices. The furan levels induced by irradiation at doses that are sufficient to inactivate 99.999% of common forborne pathogens were comparable to those found in the FDA survey. Our result will help federal regulatory agencies to make science-based decisions on the evaluation of petitions to allow irradiation of ready-to-eat foods. Action Plan Component(s) 2.3.1.4. Milestone 2.

List other significant accomplishments, if any.
Anti-technology and globalization groups opposed to food irradiation have maintained that 2-dodecylcycloutanone (2-DCB), a chemical found at trace levels only in irradiated foods, is mutagenic and that irradiated foods are therefore carcinogenic. To address this 2-DCB, using an FDA recommended test, was tested for the ability to cause mutations in human cells (TK6 lymphoblasts). The chemical did not cause mutations in human cells. Results of the studies are now being distributed as part of the USDA FNS National School Lunch Program for irradiated ground beef in order to provide school district administrators and parents with accurate science-based factual information on the toxicological safety of irradiated foods. Action Plan Component(s) 2.3.1.4. Milestone 2.

Contamination of ready-to-eat and heat-and-eat sandwiches with pathogenic bacteria has regularly been associated with food-borne outbreaks. Five common foodborne pathogens including Salmonella spp., Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157:H7, and Yersinia enterocolitica were inoculated onto heat-and-eat products including precooked cheeseburgers, veggie burgers, and a frankfurter on a bun. The results show that a radiation dose of 1.2 kGy can inactivate 2 to 4 logs of the pathogenic bacteria on each of the food products, and that a radiation dose of 2.4 kGy can inactivate 4 to 9 logs of the various pathogenic bacteria. This results can be used by regulatory agencies to evaluate a petition to allow irradiation of ready-to-eat foods, and by the food processing industry to provide safer foods for consumers. Action Plan Component(s) 2.3.1.4, 2.6.1.1. Project Plan Milestone(s) 1, 2 and 3. Milestone 1.

"Biosensor Processes for Detecting Pathogenic Bacteria in Foods"

"New Method to Detect Viable Escherichia coli O157:H7. Often it is desirable to know whether detected pathogens are alive or not. This viability test may be used as a practical guide for regulatory agencies and food processors to realistically evaluate the potential health threat of contaminated foods. While there are many new sensitive biosensor processes for pathogen detection, means to indicate the viability of target pathogens are still lacking. Thus, we developed a new luminescence process to detect viable E. coli O157:H7 in ground beef. The process involved steps to first detect the presence of E. coli O157:H7 followed by the viability test of detected E. coli. Magnetic beads coated with anti-E. coli O157:H7 antibodies were used to capture the bacteria. The identity of captured bacteria was further determined by the use of other E. coli O157:H7 specific antibodies. The captured E. coli O157:H7 was also treated with menadione, a chemical that can monitor the cellular content of nicotinamide adenine dinucleotide phosphate (NAD[P]H), a cellular viability indicator. The developed process allowed the use of the same instrumentation and the same chemistry principles for both steps. The obtained result is useful for researcher and/or engineers to design cost-effective detection processes that can generate multifaceted information of the pathogen using a single detection platform.

List other significant accomplishments, if any.
Essentials for Minimizing non-Specific Binding of Bacteria. The contamination of foods with pathogenic bacteria (e.g., Salmonella or E. coli O157:H7) may lead to substantial food poisoning epidemics. The level of pathogenic bacteria in foods is usually very low relative to the concentration of non-pathogenic bacteria. Thus, methods such as immunomagnetic beads (IMB) applications for extracting pathogenic bacteria by specific antibody reactions from foods also capture these benign bacteria by non-specific interactions thereby limiting the functionality of IMBs. Therefore, it is desirable to search for chemicals or their formulation that can minimize the attachment of non-pathogens (e.g., blocking agents or BA) to IMBs. To develop generally-useful BAs, we need to understand the processes involved in non-specific bacteria attachment to capture surfaces. Our preliminary data indicated that fluorescence polarization (FP) technique may be used to define the mode of interaction of these BAs with IMBs and bacterial surfaces. This information may be of use in improving the function of BAs.

Food-borne non-pathogenic E. coli Growth Dependent on Initial Concentration. In the normal course of working with a food-borne (chicken) E. coli isolate which sticks to IMBs (this process involves growing bacteria in 96-well or microtiter plates) we found that the distribution of population size doubling times is dependent upon the initial cell concentration. This observation suggests that some food-borne (background) organisms may communicate with each other in order to, presumably, coordinate growth under appropriate conditions. We also showed that the average doubling times from such plate assays was the same as those obtained using total aerobic plate counting (TAPC). This finding was surprising inasmuch as TAPC involves sampling from well-oxygenated media. Questions of bacterial growth media oxygenation were investigated and we found that the rate of oxygen consumption was greater than the rate of uptake even in vigorously shaking cultures until early stationary phase was reached. The latter finding is true even though the rate of oxygen uptake per cell drops between mid-log phase to early stationary phase whereupon the normalized rate of uptake levels off. These findings indicate that bacterial growth in microtiter plates is roughly equivalent to growth in flasks.

Optimized Detection of L. monocytogenes. Listeria monocytogenes is one of the leading causes of deaths related to foodborne illness and methods for the detection of the organism have been hampered by the lack of a specific antibody for its detection. Using bacterial phage display technique, we recently isolated an antibody fragment that binds specifically to L. monocytogenes and doesn’t cross-react with other species of Listeria, or any other bacterium tested. Nevertheless, a few strains of L. monocytogenes were not detected by this antibody fragment. We found that the antibody fragment binds to a target protein, ActA on the surface of L. monocytogenes. As determined by ELISA, the quantity of ActA in L. monocytogenes is affected by the environmental factors such as growth temperature and media composition. Inclusion of activated charcoal in the growth media significantly increases ActA target protein (as measured by ELISA and western immunoblot analyse) and allows the detection of all strains of L. monocytogenes by the isolated antibody fragment. Although the mechanism of ActA overexpression in the presence of activated charcoal is not completely understood, other researches have shown that it may result from the increased expression of PrfA (a positive regulatory factor affecting the expression of ActA and other L. monocytogenes virulence factors) through the binding of inhibitory compounds present in the media. This development affirms the potential of using the new antibody fragment for the detection of L. monocytogenes.

Genetic Systems for Generating Antibody Fragment Protein Fusions for Pathogen Capture and Detection. In order to be used as reagents for the detection of pathogens antibodies are chemically modified to allow them to capture and detect pathogens from complex mixtures such as food. Antibody fragments isolated by antibody phage display offer a significant advantage over traditional methods of antibody production in that the gene encoding the antibody fragment is also isolated. This allows the direct genetic fusion of the gene encoding the antibody fragment with genes encoding reporter proteins, alleviating the need to chemically modify the antibodies. We previously developed a system for generating biotinylated antibody fragments in a genetically modified E. coli. Using this system we generated biotinylated antibody fragments using the anti-L. monocytogenes antibody fragment described above, coupled the biotinylated antibody fragments to immunomagnetic beads and demonstrated their effectiveness in capturing L. monocytogenes. We have now constructed plasmid DNA that will allow the fusion of antibody fragments to bacterial alkaline phosphatase (BAP) and have designed a strategy and acquired the molecular tools to construct a plasmid DNA for the fusion of antibody fragments to green fluorescent protein (GFP). The construction of BAP and GFP fusions to antibody fragments will allow the synthesis of immunoreagents for the detection of foodborne pathogens by enzyme linked immunosorbant assays (ELISA) and fluorescence-based assays, respectively. "